The Interleukin-1 (IL-1) pathway is a cellular signaling pathway is that plays a crucial role in the mammalian inflammatory response. Several different receptors and ligands are involved in this pathway, including the ligands IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), and two IL-1 receptors referred to as IL-1 receptor Type I (IL-1RI) and IL-1 receptor Type II (IL-1RII); a soluble form of the latter also exists. Of these, it appears that IL-1RI is the signaling receptor, whereas IL-1RII does not transduce signal to a cell, but instead may be involved in regulating an IL-1-mediated response (Colotta et al., Immunol. Today 15:562; 1994). Signaling via the IL-1 pathway is complex, requiring a number of accessory molecules in addition to IL-1RI, including a receptor-associated kinase (IRAK). A serine/threonine kinase with homology to IRAK, referred to as Pelle, is found in Drosophila (for review, see Belvin and Armstrong, Annu. Rev. Cell Dev. Biol. 12:393; 1996). Another Drosophila protein, Pellino, has been reported to interact with Pelle (Grosshans et al., Mech. Dev. 81:127; 1999).
Dorsal-ventral polarization in Drosophila embryos depends upon the establishment of a gradient of nuclear localization of the Rel-like transcription factor Dorsal. The transcriptional program mediated by Dorsal results from a signaling cascade triggered by binding of an extracellular ligand Spaetzle to its receptor Toll. Intermediates of this signaling cascade include the adaptor protein Tube, the serine/threonine kinase Pelle, and Cactus, a cytosolic binding partner of Dorsal. Signals transmitted by Toll result in the degradation of Cactus, and thereby permit the nuclear importation of Dorsal. The similarity between the cytosolic domains of Toll and the mammalian interleukin-1 receptor IL1-R1 was first noted by Gay and Keith (Gay, N., and Keith, F., 1991, Nature 351: 355-356), and the number of proteins which contain the homologous regions, called the Toll/IL-1R (TIR) domain has subsequently been extended to include a larger family of receptors and intracellular signaling molecules from a variety of organisms. Those with leucine-rich repeats in their extracellular domains are broadly involved in inmate immune responses and include at least ten mammalian toll-like receptors (TLRs) which initiate inflammatory responses to microbial pathogens such as peptidoglycan, bacterial lipopeptides, bacterial lipopolysaccharides, zymosan, CpG DNA, flagellin, lipoteichoic acids, and Respiratory Syncytial Virus proteins; and plant proteins such as the N resistance gene product which mediate disease resistance. Furthermore, it is now clear that an important function of Toll signaling in adult Drosophila is in controlling responses to fungal infections.
Downstream components of the Toll signaling pathway have also been evolutionarily conserved in mammalian TLR and interleukin-1 receptor signaling pathways which culminate in nuclear translocation of the transcription factor Nuclear Factor kappa B (NF-kB). Protein kinases of the IRAK family, close homologues of Pelle (such as IRAK and IRAK4), are recruited to the activated IL-1R or TLR receptor complexes through the adaptor protein MyD88 and undergo autophosphorylation reactions. Although MyD88 is not a strict analog of Tube, both proteins contain a so-called death domain, and Tube likely serves to mediate signal transmission between Toll and Pelle, to which it binds. IRAK subsequently interacts with another adaptor molecule TRAF6, which is homologous to the recently described D-TRAF. Signals downstream of TRAF6 appear to be divergent, and not all of them are fully understood, but one consequence, in mammalian cells, is the activation of the IkB kinase (IKK) complex which directly phosphorylates the inhibitory Cactus homolog IkB at two N-terminal serine residues causing its ubiquitination and degradation. Released from a cytoplasmic association with IkB, NF-kB migrates into the nucleus. Recently, a candidate for an additional intermediate in Tube-Pelle interactions was found by yeast two-hybrid screening with Pelle as a bait sequence. This protein, called Pellino, was shown to interact with catalytically-competent Pelle, but not with a mutant form of Pelle that lacked kinase activity. Although a function for Pellino was not addressed in this study, it was suggested that it could either stabilize the activated form of Pelle, or mediate an interaction with downstream Pelle substrates.
IL-1 and other pro-inflammatory cytokines have been implicated in a variety of diseases and conditions, including rheumatoid arthritis, multiple myeloma, osteoporosis, endotoxemia and sepsis, osteoarthritis, inflammatory bowel disease, and allergy. Inhibition of the signaling of IL-1 using soluble forms of IL-1Rs, and the IL-1ra, have been shown to be useful in treating or ameliorating disease characterized by excess levels of IL-1 (Rosenwasser, J. Allergy Clin. Immunol. 102:344; 1998). Other parts of the IL-1 signaling pathway and other pro-inflammatory MAP kinase-activated pathways have also been the target of attempts to identify additional molecules that can be used therapeutically to intervene in conditions related to IL-1 and pro-inflammatory cytokines generally. Thus, there is a need in the art to identify novel molecules involved in the IL-1 and MAP kinase-activated pro-inflammatory signaling pathways, both as tools with which to investigate cell signaling and for use in identifying inhibitors of pro-inflammatory signaling. Of particular interest are novel polypeptides that are involved in stimulation of multiple pro-inflammatory signaling pathways, as inhibition of such polypeptides would more effectively inhibit inflammatory effects than inhibition of a pathway-specific polypeptide.